HBV X基因对成人肝细胞HL7702线粒体功能影响.docVIP

HBV X基因对成人肝细胞HL7702线粒体功能影响 　作者：林纳, 李 丹, 陈红英, 陈治新, 王小众 【摘要】 目的: 研究HBV X基因与线粒体COXⅢ基因相互作用以及对线粒体功能的影响。 方法: 将HBV X基因真核表达载体pCDNA3X及空载体pCDNA3转染成人肝细胞HL7702细胞, 经过2周G418筛选, 获得稳定表达的新细胞株, 分别命名为HL7702/HBx和H7702/pcDNA3, RTPCR和Western blot方法证实HBV X基因在HL7702/HBx细胞内的稳定表达。抽提细胞总RNA进行半定量RTPCR及分离细胞线粒体进行Western blot检测COX Ⅲ 表达水平变化, 并通过酶动力学方法检测线粒体细胞色素氧化酶活性。结果: 重组质粒pcDNA3X转染后经G418筛选, RTPCR和Western blot分析结果显示HL7702HBx细胞能够稳定表达HBV X基因。RTPCR和Western blot结果发现HBV X基因下调COX Ⅲ 蛋白表达而不影响mRNA水平表达。酶动力学检测发现线粒体细胞色素C氧化酶活性降低。结论: HBV X基因通过转录后水平调节COX Ⅲ表达, HBx通过与COXⅢ相互作用抑制线粒体细胞色素氧化酶活性。 【关键词】 HBx; COX Ⅲ; 线粒体细胞色素氧化酶 ［Abstract］ AIM: To study the effect of HBx on a new HBxinteracted protein COXⅢ and mitochondria. METHODS: A recombinant eukaryotic expression plasmid pcDNA3X was constructed, then pcDNA3X and pcDNA3 were steadily transfected in HL7702 separately, named HL7702/HBx and HL7702/pcDNA3. The expression of COXⅢ in HL7702/HBx, HL7702/pcDNA3 and HL7702 cells were detected by RTPCR and western blot. Mitochondrial cytochrome c oxidase activities of three groups were assayed spectrophotometrically at 550 nm by using partially purified mitochondria. RESULTS: COXⅢ mRNA expression didn’t change, while protein expression is downregulated in HL7702/HBx. Cytochrome c oxidase activity was much lower in HL7702/HBx compared with HL7702 and HL7702/pcDNA3 cells. CONCLUSION: HBx can downregulate COXⅢ expression through posttranscriptional level and inhibit mitochondrial cytochrome c oxidase activity. ［Keywords］HBx; COXⅢ; cytochrome c oxidase ［摘 要］ 目的: 研究HBV X基因与线粒体COXⅢ基因相互作用以及对线粒体功能的影响。 方法: 将HBV X基因真核表达载体pCDNA3X及空载体pCDNA3转染成人肝细胞HL7702细胞, 经过2周G418筛选, 获得稳定表达的新细胞株, 分别命名为HL7702/HBx和H7702/pcDNA3, RTPCR和Western blot方法证实HBV X基因在HL7702/HBx细胞内的稳定表达。抽提细胞总RNA进行半定量RTPCR及分离细胞线粒体进行Western blot检测COX Ⅲ 表达水平变化, 并通过酶动力学方法检测线粒体细胞色素氧化酶活性。结果: 重组质粒pcDNA3X转染后经G418筛选, RTPCR和Western blot分析结果显示HL7702HBx细胞能够稳定表达HBV X基因。RTPCR和Western blot结果发现HBV X基因下调COX Ⅲ 蛋白表达而不影响mRNA水平表达。酶动力学检测发现线粒体细胞色素